Cholangiocytes are targets for a group of human diseases termed the cholangiopathies. In vivo morphologic studies suggest that apoptosis is an important mode of cell death for many of these cholangiopathies. To date, there has been a paucity of information regarding apoptosis in cholangiocytes, due in part to the lack of a reliable and reproducible in vitro model. I have recently developed an in vitro model of apoptosis in rat and human cholangiocytes and have begun to use an array of biochemical, morphological, and cell and molecular biological techniques to define the regulatory and mechanistic aspects of cholangiocytes apoptosis. Because cholangiocytes are professional ion transporters, and because proteases have been shown to be key in mediating apoptosis, we HYPOTHESIZE that perturbations in intracellular ion homeostasis in cholangiocytes activate a signaling cascade involving proteases leading to apoptosis. The SPECIFIC AIMS for this proposal are: 1) To test the hypothesis that perturbations of intracellular ions initiates a signaling cascade culminating in apoptosis of cholangiocytes; 2) to test the hypothesis that activation of the ICE family proteases is a mechanism of causing apoptosis in rat cholangiocytes; 3) to test the hypothesis that alterations of protein levels of the Bcl-2 family of proteins regulate apoptosis in human cholangiocytes. Our long-term goals are to provide a better understanding of apoptosis in cholangiocytes during normal and diseased states, and to provide a framework for the development of therapeutic strategies for the cholangiopathies, a group of malignant, infectious, and immunological liver diseases.